Bussola, M.; Perozeni, F.; Meloni, M.; Pivato, M.; Zaffagnini, M.; Ballottari, M.

The Calvin-Benson-Bassham (CBB) cycle is the metabolic pathway responsible for CO2 assimilation in oxygenic photosynthetic organisms. Two key rate-limiting steps in this cycle are catalyzed by the enzymes fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase), making them promising targets for genetic enhancement to improve carbon fixation. A potential strategy involves overexpressing a cyanobacterial dual-function FBP/SBPase, which catalyzes both reactionsOverexpression of this enzyme in tobacco plants or in other plants led to an increase in growth rate and biomass accumulation. Here, the overexpression of the same enzyme was achieved in Chlamydomonas reinhardtii. The recombinant cyanobacterial FBP/SBPase isolated from C. reinhardtii exhibited the expected catalytic activity, being Mg2+ dependent and strongly activated in the presence of a reducing agent. The FBP/SBPase expressing lines exhibited an increased photosynthetic activity at the cell level and decreased production of singlet oxygen upon exposure to high irradiances, suggesting improved capacity to manage high excitation pressure of the photosynthetic apparatus. Increased cell volume was measured in FBP/SBPase-expressing lines under different growth conditions. However, increased growth and biomass productivity were observed only in mixotrophy when light and CO2 were limiting, leading to increased starch, protein, and lipid content on a cellular basis. This phenotype caused an increased sedimentation rate in the transformant lines: the expression of FBP/SBPase enzyme could thus be considered as a strategy to improve the cell harvesting process. These findings provide new insights into carbon metabolism in microalgae, and could, in the future, support improved biomass accumulation, paving the way for effective domestication and industrial use.